BIOLOGY 685, BIOMEDICAL TRACERS

Spring Term 2009

Instructor: Kenneth L. Campbell, Professor of Biology
M-3-322, 617-287-6676, Kenneth.campbell@umb.edu
Office Hours, M & F, 11:00 AM -1:00 PM or by appointment
Lecture/Seminar, W-1-012, M 4:00 – 7:00 PM
Description
A seminar and lab course in biomedical tracers, describing the types and uses of physical,
chemical, and biochemical tracers in the biomedical sciences. Coverage includes theory and
applications of various tracers (radioisotopes, immunoglobulins, lectins, enzymes, chromogen
labels, spin labels, heavy isotopes and particles), instrumentation for their detection and
general methods. The laboratory will include projects chosen by the student and the
instructor. Format: 2-3 seminar hours and 6 lab hours per week.
Course Philosophy
Many of the jobs currently available in academia, government and industry require knowledge
and experience with specific approaches, methodologies and/or techniques. Not infrequently
the overall training an individual possesses is of only secondary importance in obtaining a job
or position; the needs of the employer take precedence. The importance, then, of gaining
knowledge and experience with a spectrum of technologies and techniques becomes most
apparent during the search for employment. In addition, the value of realizing the existence,
possibilities, and limitations of multiple marking and tracing strategies that can be used in an
immense number of biological and biomedical contexts is enormous. Such information can
also be applied in a multitude of ways during graduate or medical training in a vast number of
programs.
This course is meant to present several of the methodologies frequently used in studies in
biology and medicine in as practical a way as possible given the size of the class and their
diversity of interests. Emphasis in the seminar will be placed on:
1. outlining the essential physical-chemical background of each of the several tracers
discussed;
2. describing the materials and instrumentation used in studies of each tracer;
3. considering practical aspects of use of each tracer, including analysis of data obtained;
and,
4. illustrating tracer use by means of examples.
Emphasis in the laboratory will be on application of one or two of the techniques to specific
focused projects chosen by the student in consultation with the instructor and any other
appropriate faculty consultants, e.g., the student's thesis mentor.

Schedule

02/02/09 4:00-7:00 Organization, Scheduling, Logistics, Introductions

Session 1 Radioisotopes
Session 2 Chromogen Labels (Absorbance, Spectrometry)

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02/16/09 President’s Day Holiday
Session 3 Fluorescence, Luminescence & Phosphorescence
Session 4 Immunoglobulins
Session 5 Immunoglobulins, Lectins & Other Binding Agents
Session 6 Enzymes
03/14-22/09 Spring Break
Session 7 Particle Labels & Quantum Dots
Session 8 Spin Labels (ESR, NMR)
Session 9 Mass Labels (Mass Spectra)
Session 10 Density Labels (X-ray, Ultrasound)
04/20/09 Patriot’s Day Holiday
Session 11 DNA Labels, Nucleotides, Modifiers, Proteins
Session 12 Polymerase Chain Reaction, End Labeling, Nick & Replacement, etc.
Session 13 Sequencing (Generations 1-3) & Microarrays
Session 14 Ionic Labels
05/13/09 Grant proposal and laboratory notebook and progress report must be in.

Notes:

1. Anticipate spending an average of not < 9-12 hrs on the course each week throughout
the term, i.e., 6-9 hrs in the library, on the web and/or in lab for each week of the term.
2. No single text covers everything in the course. Five texts are suggested for use in the
course: Guy & Ffytche, Introduction to the Principles of Medical Imaging, 2005,
covers X-ray, tomography, MRI, medical radioisotopes, and ultrasound; Manz, Pamme,
and Iossifidis, Bioanalytical Chemistry, 2004, covers instrumental and molecular
methods applied to DNA and proteins; Kessler, Nonradioactive Analysis of
Biomolecules, 2000, covers most protein or DNA label methodologies including organic
molecules and antibodies as well as PCR; Chandler and Roberson, Bioimaging:
Current Techniques in Light & Electron Microscopy, 2008, covers most microscopy
methods; and, Kricka, Nonisotopic DNA Probe Techniques, 1992, covers label
incorporation technologies upon which DNA-labeling and sequencing are based.
(Although you may choose not to purchase all of them, these books should also prove
valuable references to anyone staying in biology or biomedical sciences as a career.) In
addition, an extensive series of texts treating the topics to be covered has been provided
to course registrants. Many of these are available either in the University Library or in
Dr. Campbell's laboratory. Do not fail, however, to make use of journals (e.g., Science,
Nature, Analytical Chemistry, Analytical Biochemistry, Immunology, Trends in
Biotechnology, Biotechniques, Nature Methods), review series (e.g., Methods in
Enzymology, Progress in Biochemistry, Annual Review of Immunology, Nucleic
Acids Research), or other methods compendia that can be found in our library or in
other libraries in the Boston area, especially those at Tufts Medical School, MIT, Boston
University, Northeastern or Harvard-Countway (probably the best source, but costs $$ to
get access unless you have a Harvard affiliation). Additional information can be obtained
from many of the science faculty at UMass Boston, including Dr. Campbell's library (with
permission only), from supply catalogs such as those produced by Amersham, New
England Nuclear, Biorad, Pharmacia, In Vitrogen, Pierce Chemical, New England
Biolabs, or Promega or by use of computerized searches of such databases as Medline
or Medlars which are available through the UMass Boston library.

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 Course Requirements

1. Attendance and participation in class and laboratory (10% of grade) and, whenever
possible, in Biology Departmental Seminars.
2. A take-home final exam, extended paper, or grant application preparation (decided
upon during the first meeting of the class) will determine 90% of the grade. Any exam or
grant forms will be given out during the first three weeks of classes. Any exam, paper, or
grant application will be due no later than May 13, 2009 (the end of classes for the term).
Grants will use NIH or NSF-format and will focus on a problem chosen by you and Dr.
Campbell that makes use of one or more of the tracer techniques described in the
course. The intent of the grant preparation exercise is to expose each participating
student to the actual written instruments utilized in underwriting laboratory science and to
deepen the student's knowledge of one or more techniques to the point of being able to
prepare a competing project proposal involving the background, project design
considerations, and analytical information normally necessary to actually apply that
technology in the research laboratory. Students may work together on generation of
materials needed to fully support an application and should seek advice and answers to
questions as they arise both from Dr. Campbell and from other Biology Faculty. Students
may use whatever written materials are available in preparing an exam, application, or
paper so long as they cite those materials appropriately. The "exam" is designed to
require approximately 8-15 pages as a response; the section in grants on Preliminary
Studies/Results should incorporate a progress report on the laboratory project conducted
in connection with the course. You will have the option of considering whether to submit
the final product to a granting agency if you write a grant proposal. Papers will use the
type of format appropriate for theses and other formalized technical writing. This can be
done in conjunction with normal thesis or final project report preparation.
3. Lab project. If you are working on a Master's or independent research project already,
the most desirable projects for this course will be those incorporating one or two of the
techniques mentioned into some phase of the ongoing project. This should be done in
consultation with me and the project mentor and should be focused enough to be
accomplished with the one semester time-frame.

Medical Restrictions

If students have particular medical restrictions or conditions that require accommodation

please provide the instructor with appropriate information or forms to allow the
accommodation to be made. These should be available at the Ross Center for Disability
Services (M-1-401) at (617) 287-7430. Female students should be particularly cautious
about projects involving radiation, reactive chemicals, or biologically active materials if they
are pregnant or are intending to become pregnant during the course of the term. There are
ample ways to engage in appropriate projects that minimize these risks.

Laboratory Rules and Regulations

In the lab portion of this work all students are expected to abide by the rules of the individual
labs they may be working in. Minimally, these include observation of all safety and
housekeeping regulations, including appropriate disposal of any laboratory wastes generated
as governed by the rules of the Office of Environmental Health and Safety. If your work

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involves use of radioisotopes, you need to be given specific training in safe use of
radioisotopes (some of this is redundant on the coverage of this course) and to observe all
rules regarding use, transport, and disposal of waste isotopes. This can be handled in
coordination with the Campus Radiation Safety Office and Radiation Safety Committee. If
your work involves use of materials derived from animals other than humans, you need to
undertake a short, free, web-based course on the safe and humane use of animals which is
offered through the regulatory function of the Office of Research and Sponsored Projects
(ORSP); check the ORSP website for details. Should the work extend beyond what is
already covered by existing animal use protocols, a new animal use protocol must be filed
and approved by the Institutional Animal Care and Use Committee (IACUC) before you can
begin work. Finally, if your work involves materials derived from humans, you must
undertake a brief, free, web-based training on use of human subjects in research. Again this
is made available via the ORSP website and is overseen by the regulatory function of the
ORSP. If the work extends beyond what is already covered by existing human use protocols,
a new human use protocol must be filed and approved by the Institutional Review Board on
human use (IRB) before work can proceed.

Student Code of Conduct

All students should be familiar with the student code of conduct described in the Graduate
Bulletin or departmental graduate handbooks:
http://www.umb.edu/students/student_rights/ug_academic_regulations.html.
All exams, written materials, and electronic files containing data or written descriptions or
summaries are subject to rules regarding plagiarism and observation of copyrights; violation
of these rules may result in loss of grades and/or disciplinary actions up to and including
expulsion from Departmental Programs or the University. If there are questions regarding
use of published materials they should be brought to the instructor before submission of any
work containing copies of material derived from such published material.

Rough Outline of Topics Covered

Tracers: Types, uses, theory and application; general analytical considerations (Manz,
Pamme, and Iossifidis, Bioanalytical Chemistry, 2004; Wang and Wu, Biomedical Optics:
Principles and Imaging, 2007; Campbell and Wood, Appendix to Wood, 1994, Dynamics of
Human Reproduction: Biology, Biometry, Demography; Campbell, Quantitative
Endocrinology, manuscript; training web sites linked to PowerPoint presentations)

Radioisotopes: Radiation and nuclear medical imaging (Guy & Ffytche, An Introduction to
the Principles of Medical Imaging, 2005, nuclear medicine sections; Shung, Smith & Tsui,
Principles of Medical Imaging, 1992; Prince & Links, Medical Imaging Signals and
Systems, 2005; Cember & Johnson, Introduction to Health Physics, 2008; training web
sites linked to PowerPoint presentations; Radiation Safety Committee Information from Dr.
Campbell)

• Properties of radioactive isotopes commonly used

• 3
H, 14C, 32P, 35S, 125I
• Decay processes including energetics
• Safety issues: time, distance, shielding

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 • Radiation detectors, operation and limitations
• Scintillation counters -- liquid, solid
• Ionization detectors -- G-M, film, electron capture
• Quenching
• Statistics
• Labeling methods: synthesis, conjugation, substitution
• Isotope effects

Chromogen labels: Chemistry and visualization (Manz, Pamme & Iossifidis, Bioanalytical
Chemistry, 2004; Kessler, Nonradioactive Analysis of Biomolecules, 2000; Chandler &
Roberson, Bioimaging: Current Techniques in Light & Electron Microscopy, 2008; Wang
& Wu, Biomedical Optics: Principles and Imaging, 2007; Kricka, Optical Methods: A
Guide to the “-escences”, 2003; Hof, Hutterer & Fidler, Fluorescence Spectroscopy in
Biology: Advanced Methods and their Applications to Membranes, Proteins, DNA and
Cells, 2005; Rochow, An Introduction to Microscopy by Means of Light, Electrons, X-
Rays, or Ultrasound, 1995; Pawley, Handbook of Biological Confocal Microscopy, 2006;
Gosling, Immunoassays: A Practical Approach, 2000; Collins, Alternative Immuno-
assays, 1984; Williams, Autoradiography and Immunocytochemistry, 1980; Work & Work
Series; Van Dyke, Bioluminescence and Chemiluminescence. Instruments and
Applications. Volumes I & II, 1985; Albertson & Haseltine, Non-Radiometric Assays:
Technology and Application in Polypeptide and Steroid Hormone Detection, 1988; In
Vitrogen (formerly Molecular Probes) Catalogs & web links)

• Light and UV labels

• Spectrophotometers and colorimeters
• Aromatic compounds
• Fluorescence and phosphorescence
• Theory
• Detection -- wavelength resolution, time-resolution
• Fluorimeters
• Microscopes
• FACS
• Luminescence & luminometers
• Reduction of background, maximization of S/N ratio

Immunoglobulins & Lectins -- Affinity Labels: Production, characterization, modification

and application of biologically-derived affinity labels (Manz, Pamme, and Iossifidis,
Bioanalytical Chemistry, 2004; Kessler, Nonradioactive Analysis of Biomolecules, 2000;
Chandler and Roberson, Bioimaging: Current Techniques in Light & Electron
Microscopy, 2008; Moran, Immunotechnology: Principles, Concepts and Applications,
2006; Gosling, Immunoassays: A Practical Approach, 2000; Wu, Quantitative
Immunoassay: A Practical Guide for Assay Establishment, Troubleshooting, and
Clinical Application, 2000; Tijssen, Practice and Theory of Enzyme Immunoassays,
1985; Work & Work Series; Biorad & Pharmacia catalogs; Collins, Alternative Immuno-
assays, 1984; Hurrell, Monoclonal Antibodies: Techniques and Applications,1982;
Williams, Autoradiography and Immunocytochemistry, 1980)

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 • Structures and general properties
• IgG, IgM, IgA, IgE, IgD, MAbs
• Protein A, Protein G, Biotin-Avidin, Con A, WGA, etc.
• Affinity matrices
• Purification of IgGs and IgMs
• Use of antibody fragments
• Antigen-antibody techniques
• -- biochemical: RIA, EIA, IRMA, ELISA, FIA
• -- histochemical: PAP complex, ABC complex, adsorbed lectins, chromogen labeled
antibodies, lectins, particles; tracing on blots and gels
• Fixation techniques, washing
• Background suppression, controls

Enzymes: Purification, characterization, conjugation and applications (Manz, Pamme &

Iossifidis, Bioanalytical Chemistry, 2004; Chandler & Roberson, Bioimaging: Current
Techniques in Light & Electron Microscopy, 2008; Gosling, Immunoassays: A Practical
Approach, 2000; Tijssen, Practice and Theory of Enzyme Immunoassays, 1985; Work &
Work Series; Collins, Alternative Immunoassays, 1984; EM Series; Zojda, Enzyme
Histochemistry, 1979; Lewis, Staining Methods for Sectioned Material, 1977)

• Characteristics for use as tracers

• Conjugation & activity (Km, Vm, osmol, temp., pH)
• Clearing zone -- hydrolysis
• Metal deposition
• Change in local ionic composition
• Production of radioactive products
• Production of luminescence (e.g., luciferase)
• Availability of "delaying reagents"

Particles: Characteristics, formation and application (Chandler & Roberson, Bioimaging:

Current Techniques in Light & Electron Microscopy, 2008; Pawley, Handbook of
Biological Confocal Microscopy, 2006; EM Series by Hayat or Petruscz and Bullock;
Pharmacia and Biorad literature; Bangs Scientific literature; Nanoprobes literature; In
Vitrogen -- Quantum Dots)

• Gold, silver colloids

• Latex spheres, colloidal dyes
• Viruses, bacteria, yeast & red blood cells
• Hemocyanin, ferritin, thyroglobulin
• Quantum dots
• Radioactive spheres, flow tracing
• TEM, SEM, light detection systems
• Fluorescence detection systems

Mass detection systems: Use of non-radioactive isotopes, mass spectrometry, NMR, MRI,
tomography (Guy & Ffytche, An Introduction to the Principles of Medical Imaging, 2005,

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MRI section; Manz, Pamme, and Iossifidis, Bioanalytical Chemistry, 2004; Shung, Smith &
Tsui, Principles of Medical Imaging, 1992; Prince & Links, Medical Imaging Signals and
Systems, 2005)

• Heavy Isotopes and Spin Labels

• Mass spectrometry, gradient centrifugation, gel diffusion
• 15
N, 2H
• Disruption of naturally occurring isotope ratios: 35,37Cl, 39,41K, 16,18O, 14,15N
• Probe of chemical fine structure

Density: X-ray, SEM, ultrasound: Exploitation of variations in biological composition and

energy reflectance (Guy & Ffytche, An Introduction to the Principles of Medical Imaging,
2005, MRI section; Chandler & Roberson, Bioimaging: Current Techniques in Light &
Electron Microscopy, 2008; Shung, Smith & Tsui, Principles of Medical Imaging, 1992;
Prince & Links, Medical Imaging Signals and Systems, 2005; Wang & Wu, Biomedical
Optics: Principles and Imaging, 2007; Rochow, An Introduction to Microscopy by
Means of Light, Electrons, X- Rays, or Ultrasound, 1995)

• I, Br
• Heavy metals
• Molecular structure
• Opacity, x-ray scattering, electron back-scatter
• Spin: NMR, ESR
• 15
N, 2H, 31P, 19F, N->O.
• Probes of chemical fine structure or rotation/diffusion

DNA Labels, Nucleotides, Modifiers, Proteins & Polymerase Chain Reaction, End
Labeling, Nick & Replacement, etc.: Use of enzymes and chemistry to label nucleic acids
(Manz, Pamme, and Iossifidis, Bioanalytical Chemistry, 2004; Kessler, Nonradioactive
Analysis of Biomolecules, 2000; Kricka, Nonisotopic DNA Probe Techniques,1992; Hof,
Hutterer & Fidler, Fluorescence Spectroscopy in Biology: Advanced Methods and their
Applications to Membranes, Proteins, DNA and Cells, 2005; Keller, DNA Probes, 1989;
Erlich, PCR Technology, 1989; Innis, PCR Protocols, 1990; Hames, Nucleic Acid
Hybridization, 1985; Kirby, DNA Fingerprinting, 1997; Sambrook and Russell, Molecular
Cloning: A Laboratory Manual, 2001)

• DNA labeling strategies, radiolabeled or chemically modified nucleotides

• Gels, dots, spots, slots, and blots (N, S, & W, but no E)
• PCR theory and application
• Use of PCR in probing other systems, e.g., proteins

Sequencing (Generations 1-3) and Microarrays: Counting the As, Ts, Cs, and Gs and
exploration of macromolecules by high density assays (Manz, Pamme, and Iossifidis,
Bioanalytical Chemistry, 2004; Kricka, Nonisotopic DNA Probe Techniques,1992; Hof,
Hutterer & Fidler, Fluorescence Spectroscopy in Biology: Advanced Methods and their
Applications to Membranes, Proteins, DNA and Cells, 2005; Keller, DNA Probes, 1989;
Erlich, PCR Technology, 1989; Innis, PCR Protocols, 1990; Sambrook and Russell,

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Molecular Cloning: A Laboratory Manual, 2001; Baldi, Hatfield, & Hatfield, DNA
Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling,
2002; Schena, DNA Microarrays: A Practical Approach, 1999; Selzer, Marhöfer and
Rohwer, Applied Bioinformatics: An Introduction, 2008; 454 Diagnostics literature on
pyrosequencing)

• Labels being used in arrays

• Linking chemistries
• Detection modalities
• DNA sequencing, generations 1 to 3

Ionic Probe Techniques: Chemistry at the subcellular level (Chandler and Roberson,
Bioimaging: Current Techniques in Light & Electron Microscopy, 2008; Kricka, Optical
Methods: A Guide to the “-escences”, 2003; Hof, Hutterer & Fidler, Fluorescence
Spectroscopy in Biology: Advanced Methods and their Applications to Membranes,
Proteins, DNA and Cells, 2005; Williams, Autoradiography and Immunocytochemistry,
1980)

• Ion probe methods in ultramicroscopy

• Ion-specific fluorophores
• Chelators in ion work
• Enzymatic and chromogen reactions in histochemistry